Angelman syndrome is a neurological disorder affecting ~1/15,000 people that results in severe mental retardation. The disease is caused by loss-of-function mutations in Ube3a, which encodes a HECT-domain ubiquitin ligase or E3 protein. E3 proteins are specificity factors for ubiquitination; they bind specific substrates and bring the ubiquitination machinery to them. A simple model for the biochemistry of Angelman syndrome is that loss of UbeSa activity results in overexpression of the E3's normal substrates. The Drosophila genome contains a Ube3a homolog called CG6190. The goal of the research proposed is to generate a Drosophila model for Angelman syndrome that will allow a test of this hypothesis and the identification of the substrates relevant to the disease. First, it will be determined if Drosophila CG6190 knock-out mutants have an Angelman syndrome-like phenotype. Mutant flies will be analyzed for morphological and functional defects in their central nervous system. Second, the expression pattern of CG6190 mRNA and protein during Drosophila development will be described. In situ hybridization will be used to detect mRNA in whole organisms and anti-CG6190 antibodies will be generated and used to detect the protein. Third, it will be determined if Drosophila CG6190 and human UbeSa are functional homologs. By generating transgenes, the abilities of wild-type CG6190 and human UbeSa to complement the CG6190 mutant phenotype and to generate mutant phenotypes when overexpressed will be compared. In addition, site-directed mutagenesis will be used to generate alleles of the Drosophila and human genes that contain mutations identified in Angelman syndrome patients. The effects of expressing the resulting mutant alleles will be tested similarly. Finally, a genetic screen will be performed in Drosophila to identify candidate CG6190/Ube3a substrates. Overexpression of wild-type CG6190 in the Drosophila eye results in a severe mutant phenotype. Modifiers of the eye phenotype will be isolated and the mutant genes identified.